1. Field of the Invention
The present invention is generally directed to the fields of cancer biology and biochemistry. More particularly, the present invention is directed to a method for inhibiting the formation of metastases from primary tumors growing in mammals.
2. Description of Related Art
As cancer treatments using radiation and/or chemotherapies become more effective, and more people live for longer periods of time following treatment, cancer survivors are faced with a significant risk of developing therapy-induced secondary tumors (P. Y. Leverger et al., 1998; B. E. Johnson, 1998; J. E. Karp et al., 1997; S. Bhatia et al., 1996). Because of the inherent mutagenicity of ionizing radiation and most anti-cancer drugs, investigators as early as 1978 predicted that therapy-induced secondary tumors would become a major health issue (T., Sugimura et al., 1978). This is most evident in the treatment of children and young adults having potentially curable cancers such as Hodgkin's disease. It has been reported that women, treated for Hodgkin's disease in their teens with radiation and chemotherapy, had a 75-fold increased risk for the development of breast cancer by age 40, as compared to matched control populations. The estimated actuarial cumulative probability of breast cancer development in this population was 35% (S. Bhatia et al., 1996). The issue of therapy-induced secondary cancers could be effectively addressed if novel anti-cancer agents lacking mutagenic and carcinogenic properties could be identified and developed. This approach, however, is not at present either practical or feasible. A much more reasonable approach is the addition of agents that do not affect therapeutic efficacy but do possess anti-mutagenic and anti-carcinogenic properties to standard cancer therapies involving radiation and cytotoxic drugs.
The application of agents to inhibit the carcinogenic process in cells is defined as chemoprevention (M. A. Morse et al., 1993; G. J. Kelloff et al., 1994; A. J. Alberg et al. 1998). Chemoprevention is a broad term that encompasses the prevention of cancer in high-risk disease-free individuals; inhibition of second primary tumors induced by chronic exposure to carcinogens due to high risk life styles, i.e., the field cancerization process (W. K. Hong et al., 1997); and the prevention of new cancers in patients having a good prognosis for cure but who are at risk of developing therapy-induced secondary tumors. Chemoprevention applied to the first two groups generally involves the chronic administration of low levels of anti-carcinogenic agents with the intention of inhibiting both the initiation and progression steps of the carcinogenic process (F. L. Meyskens, Jr., 1991). Application of chemoprevention to the third group is somewhat different from that of the first two groups in that patients already will have cancer, have a good prognosis, and are exposed to known doses of highly carcinogenic therapeutic agents such as ionizing radiation and chemotherapeutic drugs. In this group, the chemopreventive agent can be administered as an acute dose at the time of therapy to reduce the probabilities of initiation and early promotional events involved in the therapy-induced carcinogenic process. However, since the target population for this approach are patients having a good prognosis for cure, it is essential that the chemopreventive agents used do not diminish therapeutic efficacy or enhance tumor spread and metastases formation.
While a large number of drugs have been clinically studied, thiol-containing compounds appear to represent an effective class of agents for use in chemoprevention. The thiol N-acetyl-L-cysteine (L-NAC) has been clinically evaluated both in Europe (project Euroscan) and the U.S. (National Cancer Institute) as a chemopreventive agent capable of inhibiting carcinogenesis (S. De Flora et al., 1995) and mutagenesis (S. De Flora et al., 1985). Likewise, the synthetic dithiolthione oltipraz (4-methyl-5-pyrazinyl-3H-1,2-dithiol-3-thione) has been clinically investigated as a chemopreventive agent (A. B. Benson, III, 1993; W. Kim et al., 1997) and found to be effective in inhibiting both the initiation and progression stages of carcinogenesis (G. J. Kelloff et al., 1994). Both L-NAC (S. De Flora et al., 1985) and oltipraz (P. C. Hayes et al., 1991; T. W. Kensler et al., 1995) also were found to increase intracellular glutathione (GSH) levels in exposed cells and to independently activate nuclear transcription factor κB (NFκB) and subsequent gene expression and enzyme activity of manganese superoxide dismutase (MnSOD) (K. C. Das et al., 1995; J., Antras-Ferry et al., 1997). Amifostine (S-2-[3-aminopropylamino]ethylphosphorothioic acid; also referred to as WR-2721) is in clinical use as a cytoprotective drug to minimize radiation and/or chemotherapeutic toxicity to normal tissues in patients treated by high dose cisplatin for ovarian cancer (R. T. Dorr, 1998; J. A. Foster-Nora et al., 1997; C. M. Spencer et al., 1995). In preclinical studies, this drug and its active thiol metabolite WR-1065 (2-[{aminopropyl}amino]ethanethiol) were found to be anti-carcinogenic (L. Milas et al., 1984; B. A. Carnes et al., 1992) and anti-mutagenic (Y. Kataoka et al., 1992; D. J. Grdina et al., 1992; Y. Kataoka et al., 1996) and capable of activating NFκB and MnSOD gene expression.
Cytoprotection is the use of a chemical agent to prevent cell killing and/or loss of function in dose limiting normal tissues exposed to radiation and/or chemotherapy during the treatment of cancer. Since the magnitude of cytoprotection obtained is dose dependent, maximum tolerated doses of cytoprotective agents are routinely administered during cancer treatment for the protection of normal tissues. This approach is only appropriate if it can be shown that enhanced cytoprotection is selectively limited to only normal as compared to malignant cells and tissues. Amifostine is currently limited for use in the clinic by the FDA to only patients who are not expected to enjoy a significant survival benefit or cure from their chemotherapy (Package insert for Ethyol®, 1997; News and Product Notes, 1996). The basis for this restricted use of amifostine as a cytoprotector is based on early studies with animal models. It was demonstrated that large cytoprotective doses of amifostine could protect animal tumors to a similar magnitude as reported for normal tissues, i.e., protection factors of 1.2 to 2.5 (L. Milas et al. 1984; J. S. Rasey et al., 1986; F. A. Steward et al., 1983; M. Penhaligon, 1984; S. L. McChesney et al., 1986). It has been demonstrated that there is a threshold dose of amifostine below which no cytoprotection can be demonstrated in either normal or malignant cells (D. J. Grdina et al., 1995; Y. Kataoka et al., 1996; D. J. Grdina et al., 1999). Maximum cytoprotection in the C3H mouse system requires an amifostine dose of 400 mg/kg while doses below 100 mg/kg are completely ineffective (L. Milas et al., 1984; Kataoka et al., 1996; D. J. Grdina et al., 1999). In contrast, chemopreventive doses of amifostine required to prevent radiation-induced mutations at the hprt locus in splenocytes in these mice ranged from as low as 25 mg/kg to a high of 400 mg/kg, with the effect diminishing only at doses less than 10 mg/kg (D. J. Grdina et al., 1992). These data highlight the fundamental difference implicit in the design of studies to evaluate the use of thiols in chemoprevention as compared to cytoprotection. Chemoprevention focuses on determining the minimum concentration of thiol required to afford only prevention of therapy-induced mutations and inhibition of carcinogenic processes.
Previous applications have defined these roles for phosphorothiorates and their corresponding thiols, when administered at low and non-cytoprotective doses, in preventing radiation-induced mutations in nonmalignant cells (i.e., chemoprevention). Chemicals of the phosphorothioate genus and associated metabolites can protect against somatic mutations when administered to mammals following a mutagen exposure, as detailed in U.S. Pat. Nos. 5,567,686; 5,488,042, and 5,891,856, which are herein specifically incorporated by reference in their entirety. This protection is irrespective of the nature of the mutagenic event or source of radiational or chemical insult, as detailed in U.S. Pat. No. 5,869,338, which is incorporated by reference in its entirety.
In addition to their anti-mutagenic properties, these thiols have been reported to be effective at inhibiting tumor cell growth under both in vitro and in vivo experimental conditions. As early as 1975, it was reported that cysteamine could inhibit the growth of five different transplanted tumors in mice. The largest effect was observed when it was administered to test animals at the time of tumor-transplantation (C. A. Apffel et al., 1975). The thioglycerol (i.e., lacking a terminal amino group) and mercaptopropylamine (i.e., 3-carbon homologue of cysteamine) were significantly less effective than cysteamine while cystamine (i.e., the disulfide form of cysteamine) was completely ineffective in inhibiting tumor growth. This led to the conclusion that both active thiol and amino groups are required for the maximum inhibitory effect on tumor cell growth. The ability to inhibit tumor growth also has been reported for NAC (S. De Flora et al., 1996).
One major limitation of general cancer strategies is the inability to inhibit or eradicate metastasis. It is well recognized that metastatic spread of disease is a poor prognostic factor for the treatment of human cancer. To date, strategies designed to attack the metastases problem focus only on the development of cytotoxic drugs and treatments to kill metastases that have already escaped the primary tumor site. There exists a need for a method for protecting against the formation of metastases from primary tumors growing in mammals, irrespective of the cancer therapy, which will be amenable to pre- and/or post cancer therapy administration and which will be optimally effective at non-toxic concentrations so as to allow use in mammals and also allow for multiple, as well as single, administrations.